|Publication number||US4298685 A|
|Application number||US 06/035,619|
|Publication date||Nov 3, 1981|
|Filing date||May 3, 1979|
|Priority date||May 4, 1978|
|Also published as||DE2953449A1, EP0005271A2, EP0005271A3|
|Publication number||035619, 06035619, US 4298685 A, US 4298685A, US-A-4298685, US4298685 A, US4298685A|
|Inventors||Indu Parikh, Pedro Cuatrecasas|
|Original Assignee||Burroughs Wellcome Co.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (4), Non-Patent Citations (5), Referenced by (130), Classifications (30), Legal Events (2)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The present invention provides a quantitative method for the determination of biological substances and more particularly, provides an enzyme immunoassay of such substances in biological fluids.
There is a continuous need in medical practice and research for rapid, accurate, quantitative determinations of biological substances at extremely low concentrations. The presence of drugs or narcotics in body fluids, such as saliva, blood or urine, has to be determined in very small quantities with satisfactory accuracy. In addition, in medical diagnosis, it is frequently important to know the presence of various substances which are synthesised naturally by the body or ingested. These include hormones, both steroids and polypeptides, prostaglandins, and toxins as well as other materials which may be involved in body functions.
To meet these needs, a number of ways have been devised for analysing for trace amounts of materials. A common method for isolating and detecting substances in biological fluid is use of thin layer chromatography (TLC), for example, in combination with mass spectroscopy or gas phase chromatography. However, TLC has a number of deficiencies in being slow, being subject to a wide range of interfering materials, and suffering from severe fluctuations in reliability. Therefore, the absence of satisfactory alternatives has resulted in intense research efforts to determine improved methods of separation and identification.
An alternative to TLC has been radioimmunoassay (RIA). Here antibodies for specific haptens or antigens are employed. By mixing an antibody with solutions of the hapten or antigen, and with a radioactive hapten or antigen analogue, the radioactive analogue will be prevented from binding to the antibody to an extent directly related to the concentration of hapten or anitgen in the solution. By then separating and assaying the free radioactive analogue from the antibody bound radioactive analogue, one can indirectly determine the amount of hapten or antigen in the original solution. However, the use of radioisotopes in such an assay could be a potential health hazard and, furthermore, the instrumentation generally required for radioimmunoassay is relatively sophisticated and generally too expensive too allow small hospitals and physicians to routinely perform, for example, a patient's blood or urine analysis. Enzyme immunoassay overcomes the above problems and in addition, has the unique advantage of potential amplification of the measured parameter.
In essence this method replaces the radioactive biological substance analogue with an enzyme labelled biological substance (hapten or antigen). Such modified enzyme molecules retain their enzymatic activity and the enzyme-labelled biological substance will compete for antibody complex formation with the unknown amount of free biological substance in the system. The complexes may be separated (cf. U.K. Pat. No. 1,348,935) in view of their insolubility in certain instances and the activity of this, or the part remaining in solution is used as a measure of the amount of antigen originally present. The same principle may be applicable to a reverse system, using enzyme-labelled antibodies whenever the unmodified version of the same antibody present in biological fluids has to be determined.
It has now been found that covalent attachment of biotin (Vitamin H) to the antibody molecule, resulting in a soluble biotin-tagged complex, facilitates convenient separation of all antibody forms including all enzyme-labelled and unlabelled biological substance-antibody complexes. The separation process can then be performed by the use of the biotin-specific receptor protein, avidin, which is immobilised in an insoluble form. The very strong affinity between avidin and biotin, which approaches covalent bond character, results in insolubilisation of all antibody forms and consequently allows an extremely efficient and easy removal of all biotin-tagged antibodies, and complexes formed by such antibodies.
According to one aspect of the present invention, therefore, there is provided a process for the detection and/or determination of a biological substance in a test sample, which comprises admixing the test sample, a predetermined quantity of a soluble enzyme-labelled form of the biological substance, and a predetermined quantity of a soluble biotintagged antibody raised against the biological substance, allowing to come to equilibrium, adding insolubilised avidin, separating the resulting solid phase from the liquid phase and determining the enzyme activity of either of these phases.
For the purposes of the present invention, any biological substance may be detected and/or determined for which an appropriate antibody may be produced having satisfactory specificity and affinity for the biological substance. The recent literature contains an increasing number of reports of antibodies for an increasingly wide variety of biological substances. Compounds for which antibodies can be provided range from simple phenylaklyl amines, for example amphentamine, to very high molecular weight polymers, for example proteins.
The biological substances for detection and/or determination in the process of the present invention may be divided into three different categories, based on their biological relationship to the antibody. The first category is antigens, which when introduced into the blood stream of a vertebrate, result in the formation of antibodies. The second category is haptens, which when bound to an antigenic carrier, and the hapten-bound antigenic carrier is introduced into the bloodstream of a vertebrate, elicit formation of antibodies specific for the hapten. The third category of biological substances includes those which have naturally occurring antibodies in a living organism and the antibodies can be isolated in a form specific for the biological substance.
The most important group of biological substances for the purposes of the present invention are those of the second category, the haptens. Methods for the production of antibodies to the three different categories of biological substances are well known in the art.
Selection of the enzyme for use in the present invention is governed by a number of criteria. Thus it should possess potentially reactive groups to which the biological substance can be coupled without destroying enzyme activity and should not occur naturally to an appreciable extent in the type of tissue to be assayed for the said biological substance. In addition, the enzyme should have a relatively long shelf life, a high specific activity and also be capable of being easily assayed, for example with a visible light spectrophotometer.
Examples of enzymes which may conveniently be employed in the process of the present invention are, malate dehydrogenase, staphylococcal nuclease, delta-5-ketosteroid isomerase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase, alpha glycerophosphate dehydrogenase, triose phosphate isomerase, and horse radish peroxidase, more preferably, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase and ribonuclease. Normally it is preferred to purify the enzyme, for example by dialysis against saline, before use.
The preparation of the enzyme-labelled biological substances for use in the present invention can take place in various ways known per se in the art. Some biological substances may already possess groups that can be cross-linked with reactive groups at the surface of the enzyme, while other substances will have to be provided with such groups by organic chemical reactions. It should be emphasised that neither the original immunological properties of the biological substance nor the activity of the enzyme ought to change appreciably during this process. The groups of the enzyme which are particularly suited for coupling reactions are amino and carboxyl groups. If the modified or unmodified biological substance also possesses such groups, the coupling can be performed by, for example, reactions known from peptide synthesis. Furthermore, such substances, as for example, glutaraldehyde, difluorodinitrodiphenylsulphone, toluene diisocyanate, di- and trichloro-s-triazine and others can be employed for the coupling reaction.
Examples of the coupling of biological substances to enzymes are described in, for example, L. A. Steinberger, Immunocytochemistry, Prentice Hall, New Jersey (1974).
Specific examples of the coupling of haptens to proteins are described in, for example, C. A. Williams and N. W. Chase, Methods of Immunology and Immunochemistry Vol. 1 Academic Press, New York, 1967. The methods described are used for the preparation of conjugates for immunisation but they can also be used for the preparation of the enzyme-labelled biological substances which are essential in the process of the present invention.
Biotin-tagged antibody is conveniently prepared by reaction of a biotin derivative, for instance a biotin ester derivative such as the N-hydroxysuccinimide ester of biotin, with the antibody. The biotin ester derivative is dissolved in a polar, aprotic solvent, for example dimethylformamide, and is then added in a 20 to 300 molar excess to the antibody in 0.01 M to 1.0 M, preferably 0.05 M to 0.5 M, most preferably 0.1 M phosphate buffer, for example potassium phosphate buffer at from pH 6.5 to pH 8.5, preferably pH 7.5. After admixture of the reactants, the reaction is allowed to proceed at a temperature of from 2° to 10° C., more preferably at 4° C. for a time sufficient for its completion. Normally this takes of the order of 10 hours. After completion of the reaction, the biotin-tagged antibody may be separated from the reaction mixture by standard methods well known in the art, for example by gel permeation chromatography on, for instance, a cross-linked dextran.
Insolubilised avidin, that is, avidin immobilised by attachment to a solid support, may be prepared by a method in actual use or described in the literature, for example, by covalent binding with macromolecular insoluble carriers such as agarose, polystyrene, polyacrylamide, nylon, cross-linked dextran or filter paper or by physical coupling to insoluble carriers such as glass beads or plastic objects, or to the inside of test tubes made from either plastic or glass or to microtitre plates.
Specific examples of coupling haptens and other biological molecules to agarose and polyacrylamides are described by Cuatrecasas in J. Biol. Chem., 245, 3059-3065, (1970). W. B. Jacoby and M. Wilcheck; Methods in Enzymology; Vol. 34--Academic Press, New York 1974. These methods may also be used, in principle, to prepare (a) avidin-solid support, (b) hapten-protein conjugates and (c) biotin-antibody conjugates. Conveniently either avidin or the carrier is activated before covalent binding occurs, but most conveniently the carrier is activated before covalent binding. In one form of the invention the carrier used is agarose since this exhibits excellent coupling of avidin and retention of biotinbinding capacity. Most preferably, benzoquinone-activated agarose is used due to its ease of preparation, and its lack of non-specific absorption of enzyme. In other forms of this invention avidin is coupled to nylon rings or rods or to the inside of polystyrene test-tubes.
A schematic representation of the assay is presented in FIG. 1. To the vessel in which the reaction is to take place are added at room temperature and at near physiological pH, preferably at pH 7, successively, with a minimum time interval between the additions: free biological substances for example, contained in a serum sample, an aqueous solution of enzymelabelled biological substance, and a quantity of previously tagged antibody sufficient to neutralise (ie. complex with) 50%-80% of the enzyme-labelled biological substance. These three components are mixed, and immediately insolubilised avidin is added to the reaction mixture to create a heterogeneous system and the mixture shaken until a predetermined equilibrium point is reached. The quantity of avidin used is normally several hundred fold in excess of that theoretically required. The solid phase removes the free antibody as well as the antibody bound to the biological substance. The supernatant contains enzyme-labelled biological substance in direct proportion to the level of free biological substance, whilst the deposited solid phase has enzyme-labelled biological substance in inverse proportion to level of the free biological substance. Either the deposited solid phase or the supernatant can then be assayed for enzymatic activity to determine the amount of biological substance present in the unknown sample.
The enzyme activity measurement of the solid and/or liquid phase of the reaction mixture resulting from the process of the present invention may be carried out by methods already known in themselves. See, for example, H. U. Bergmeyer, Method for Enzymatic Analysis, Academic Press, New York (1965). The assay of the carefully separated and rinsed solid phase may take place after removal of the supernatant by, for example, aspiration.
The various forms in which the reagents of the present invention can be used are manifold. For instance, the enzyme labelled biological substance can be freeze-dried or dissolved in a buffer. Furthermore, a solid carrier for example, a strip of paper impregnated with the enzyme-labelled biological substance, can be employed.
For carrying out the process for a single test according to the present invention, use can be made of a pack comprising, in separate containers:
(a) a preselected quantity of enzyme-labelled biological substance;
(b) a preselected quantity of biotin-tagged antibody raised against the said biological substance;
(c) a preselected quantity of insolubilised avidin; and
(d) a substrate or substrates and co-factors for the determination of the activity of the enzyme employed, together with instructions to admix the enzyme-labelled biological substance, the biotin-tagged antibody raised against the biological substance, and a test sample, allowing the mixture to come to equilibrium, then adding the insolubilised avidin, separating the resulting solid phase form the liquid, and determining the enzyme activity of either of these phases using the enzyme substrate or substrates and co-factors.
If required, it may also contain the necessary auxiliaries for making a dilution series of the test sample to be examined for a quantitative determination, such as test tubes, pipettes and flasks of diluent.
The invention is illustrated further by the following examples which are not to be construed as limiting the invention to the specific procedures described in them.
Digoxin was coupled to enzyme in the following manner:
3 H-digoxin (250 μCi/mg) in ethanol was reacted with a slight molar excess of sodium metaperiodate for two hours. An aliquot of oxidised 3 H-digoxin (50 nmoles) was added to enzyme (5 nmoles) in sodium acetate buffer (1 ml; 200 mM; pH 7). The final concentration of ethanol was usually 10-20%. Sodium cyanoborohydride (10-20 mg) was added and the reaction was allowed to proceed for 3 days at 4° C. Labelled enzyme was separated from the reactants by gel exclusion chromatography on Sephadex G-50. The table below shows the degree of substitution by digoxin (calculated by means of radioactivity measurements) and the retention of enzymatic activity for four enzymes after reaction under the described conditions.
______________________________________ Digoxin substitution Specific activity (M/M) recovered as %Enzyme 3 H counting RIA of control______________________________________Malate dehydrogenase 0.50 0.10 36.7Alkaline Phosphatase 1.10 0.15 92.3Glucose Oxidase 1.70 0.30 100.0Asparaginase 1.00 0.60 100.0______________________________________
Conjugation of 3 H-digoxin with malate dehyrdogenase was investigated more fully (FIG. 2). Oxidised digoxin was reacted at various concentrations (molar excess varied from 5 to 50) with aliquots of enzyme, and the retention of enzyme specific activity was determined for each point. In addition, the degree of substitution of digoxin was determined both by tritium counting to determine the absolute number of haptens and by RIA to determine the number of immunologically reactive digoxin residues. The data shows that malate dehydrogenase is very sensitive to the degree of labelling by digoxin and that only 20% of the groups attached are functionally available to interact with antibody.
Avidin was coupled directly to a solid support of benzoquinone-activated sepharose. The avidin-gel was diluted, as desired, with underivatised sepharose.
A 50% suspension of agarose in sodium phosphate buffer (100 mM; pH 8.0) was added in a 4:1 volume/volume ratio to p-benzoquinone (250 mM) in ethanol. After stirring the suspension for 1 hour at room temperature, the activated gel was washed by suction filtration using equal volumes of, successively, ethanol (20%), aqueous sodium chloride (1.0 M), water, and sodium phosphate (pH8, 100 mM). The gel (1 volume) was then added to avidin (1 volume; 10 mg/ml) dissolved in sodium phosphate buffer (pH8, 100 mM), and the resulting suspension was shaken for 15 hours at 4° C. After this time, the avidin-gel conjugate was washed successively with 5 volumes each of sodium acetate (0.1 M, pH 4.0) containing sodium chloride (500 mM), sodium bicarbonate (0.1 M, pH 9.0) containing sodium chloride (500 mM), and water. The substitution of avidin on the gel was in the range of 4-5 mg/ml packed gel. The substituted avidin was found to have 100% of its biotin binding capacity.
Sheep antidigoxin antibody was treated variously in potassium phosphate (0.1 M; pH 7.5) with a 20 to 300 molar excess of the N-hydroxysuccinimide ester of biotin in dimethylformamide (DMF). The final concentration of DMF was 50%. After incubation overnight at 4° C., the biotinised antibody was separated from reactants by gel permeation chromatography on Sephadex G-50. The titre of the biotinised antibody was assessed using 125 I-digoxin and dextran-coated charcoal. The sensitivity of the biotinised antibody to avidin-gel was assessed using previously iodinated antibody and measuring the take-down of 125 I by avidin-gel. At levels of biotinisation above 3 biotin molecules per antibody molecule, the antibody retained its titre for digoxin and was completely absorbed by avidin-gel. The antibody-biotin preparation chosen for the enzyme immunoassay empirically met these criteria.
Glucose oxidase-digoxin conjugates, with various levels of substitution of digoxin, were titrated with various dilutions of biotinised antibody and excess avidin-gel (FIG. 3). The enzyme derivatives has been previously shown not to be inhibited by even very high concentrations of anti-digoxin antibody. The data shows that at between 1:100 and 1:1000 dilution of the stock antibody solution, the digoxin-enzyme and antibody are at approximately stoichiometric levels such that essentially all enzyme activity is precipitated by the gel. The data also shows that with increasing digoxin substitution less antibody is needed to precipitate 50% of the enzyme activity, indicating that the immunoreactivity of the enzyme conjugate is increasing with substitution.
Using appropriate levels of glucose oxidase-digoxin, biotin-tagged antibody, and excess avidin-gel, the time course to reach equilibrium between the three components of the assay was followed. Glucose oxidase-digoxin and avidin-gel were preincubated at room temperature. At t=0, biotin-tagged antibody was added and the mixture was shaken at room temperature. At various time intervals, aliquots of the assay mixture were centrifuged and the supernatants assayed for enzyme activity. In general, maximum takedown of enzyme-labelled digoxin by the antibody-avidin gel complex occured within 90 minutes, while the binding of biotin-tagged antibody by avidin-gel is essentially complete within 10 minutes.
Serum digoxin (50 μl), glucose oxidase-labelled digoxin (50 μl) which contained 1 ng/ml of bound digoxin, and appropriately diluted biotin-tagged antidigoxin antibody (50 μl) were mixed in a test tube (FIG. 1). Immediately a 50% aqueous suspension of avidin-sepharose (50 μl) which had been previously diluted 1:10 with native sepharose was added. All assay reagents were in phosphate buffer (50 mM; pH 7) containing bovin serum albumin (0.1%). The reaction mixture was then incubated for 2 hours at room temperature with shaking, and was subsequently suspended in ice-cold buffer (5 ml) containing bovin serum albumin (0.1%). The mixture was centrifuged and the supernatant aspirated away to leave the gel pellet. After washing the pellet, assay buffer (1.0 ml) containing glucose (100 mM), o-dianisidine (0.1 mg/ml) and horse radish peroxidase (7.5 μg/ml) in sodium phosphate buffer (100 mM; pH 6) was added, and shaking was performed for 1 hour. Alternatively, 0.1 mM; 2,2-azino-bis-(3-ethylbenzthiozoline sulphonic acid) (ABTS) can be substituted for the o-dianisidine as an enzyme substrate.
After this time the mixture was cooled, and the optical density of the supernatant was recorded at a wavelength of 450 nm and the values of the patient sera compared to a standard curve constructed with control sera.
FIG. 4 shows the effect of addition of various amounts of free digoxin (plotted as the logarithm of the digoxin concentration in ng/ml versus the percentage of total enzyme activity on the gel) to the incubation mixture. The relationship between free digoxin levels and the enzyme activity is linear over 0.15 to 10.00 ng/ml of free digoxin.
To illustrate the general applicability of the principle of the herein described enzyme immunoassay, the narcotic drug codeine was assayed by this method.
Codeine hemisuccinate (20 μmoles) dissolved in dry, redistilled dimethylformamide (600 μl) was added to 200 μmoles N-hydroxysuccinimide dissolved in dry, redistilled dioxane (300 μl) and 20 μmoles dicyclohexylcarbodiimide in dry, redistilled dioxane (30 μl). The mixture was allowed to react a room temperature for 5-6 hours in a tightly stoppered test tube. The codeine hemisuccinate N-hydroxysuccinimide thus prepared in situ was used without purification to couple to the enzyme. To 5 nmoles of enzyme (glucose oxidase) dissolved in 600-900 μl of sodium acetate buffer (100 mM, pH 7.0) was added 100-500 μl (preferably 300 μl) of the codeine N-hydroxysuccinimide ester solution as prepared and described above. After 12-24 hours at room temperature the enzyme (codeine conjugated together with any unconjugated enzyme) was separated from the reactants by gel exclusion chromatography. The degree of codeine substitution to glucose oxidase was determined by radioimmunoassay. As in the case of digoxin the degree of substitution of codeine varies depending on the reaction conditions. A substitution of 3-5 codeine molecules per enzyme (glucose oxidase) molecule was found to be ideal for the present assay method.
This reaction was performed as described in Example 1 for coupling of biotin to anti-digoxin antibody. The titer of biotinized antibody was essentially unaltered as compared to the underivatized antibody. Antibody preparations with 3 or more convalently bound biotin molecules were completely precipitable by solid-phase avidin and were found to be ideally suited for the present assay.
With appropriate, predetermined quantities of enzyme-codeine conjugate, biotinized anti-codeine antibody and solid-phase avidin the time course to reach equilibrium between the three components of the assay was determined. Usually after 45-60 minutes the system is at equilibrium. Codeine standard (0.1 to 10,000 ng/ml solutions (50 μl), glucose oxidase-codeine conjugate (50 μl, containing 1-20 fmoles codeine) and appropriately diluted biotin tagged anti-codeine antibody were mixed in a test tube. A 50% aqueous suspension of solid-phase avidin (50 μl) was added to the above mixture. All assay reagents were prepared in 50 mM phosphate buffer, pH 7 containing 0.1% bovine serum albumin. The reaction mixture after incubation for 2 hours at room temperature was diluted with 5 ml of ice-cold buffer containing 0.1% bovine serum albumin. The mixture was centrifuged and the supernatent aspirated away. After washing the pellet the assay buffer (1 ml) containing glucose (100 mM, o-dianisidine (0.1 mg/ml) and horse radish peroxidase (7-5 μg/ml) in phosphate buffer (100 mM, pH 6) was added. The assay mixture was incubated for 30-90 minutes (preferably 60 minutes) at room temperature, cooled in ice bath and optical density at 450 nm was recorded. Thus a standard curve with various codeine concentrations was constructed (FIG. 5).
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3839153 *||Dec 10, 1971||Oct 1, 1974||Akzona Inc||Process for the detection and determination of specific binding proteins and their corresponding bindable substances|
|US3852157 *||Nov 6, 1972||Dec 3, 1974||Syva Corp||Compounds for enzyme amplification assay|
|US4017597 *||Oct 30, 1974||Apr 12, 1977||Monsanto Company||Unitized solid phase immunoassay kit and method|
|US4134792 *||Dec 6, 1976||Jan 16, 1979||Miles Laboratories, Inc.||Specific binding assay with an enzyme modulator as a labeling substance|
|1||*||Bayer, et al. "Preparation of Ferriton-Avidin Conjugates by Reductive Alkylation for Use in Electron Microscopic Cytochemistry", J. Hist. Cytochem., vol. 24, No. 8 (1976) pp. 933-939.|
|2||*||Heggeness et al. "Use of the Avidin-Biotin Complex for the Localization of Actin and Myosin with Fluorescence Microscopy", J. Cell Biol., vol. 73 (1977) pp. 783-788.|
|3||*||Hofmann et al. "Avidin-Biotin Affinity Columns, General Methods for Attaching Biotin to Peptides and Proteins", J. Am. Chem. Soc., vol. 100, No. 11 (1978) pp. 3585-3590.|
|4||*||Manning et al. "A Method for Gene Enrichment Based on the Avidin-Biohn Interaction Application to the Drosophilia Ribosoma RNA Genes", Biochem., vol. 16 No. 7 (1977) pp. 1364-1370.|
|5||*||May et al. "NEO29-(t)-Biotinyl insulin and its Complexes with Avidin", J. Biol. Chem., vol. 253, No. 3 (1978) pp. 686-690.|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US4395486 *||Aug 19, 1981||Jul 26, 1983||Medical College Of Ga. Research Inst., Inc.||Method for the direct analysis of sickle cell anemia|
|US4444878 *||Dec 21, 1981||Apr 24, 1984||Boston Biomedical Research Institute, Inc.||Bispecific antibody determinants|
|US4496654 *||Apr 8, 1983||Jan 29, 1985||Quidel||Detection of HCG with solid phase support having avidin coating|
|US4506009 *||Mar 30, 1982||Mar 19, 1985||University Of California||Heterogeneous immunoassay method|
|US4530900 *||Sep 13, 1982||Jul 23, 1985||Seragen Diagnostics Inc.||Soluble insoluble polymers in enzymeimmunoassay|
|US4535057 *||Jul 26, 1982||Aug 13, 1985||Amf Incorporated||Immunoassay employing monoclonal herpes simplex antibody and biotin-avidin detection system|
|US4582810 *||Sep 30, 1983||Apr 15, 1986||Becton, Dickinson And Company||Immuno-agglutination particle suspensions|
|US4656252 *||Jun 4, 1984||Apr 7, 1987||Giese Roger W||Amidobiotin compounds useful in a avidin-biotin multiple layering process|
|US4684609 *||Jun 15, 1981||Aug 4, 1987||Vector Laboratories, Inc.||Method and substance for the enhanced labelling of cellular material|
|US4692403 *||Nov 28, 1984||Sep 8, 1987||The Texas A&M University System||Methods and compositions for the detection of acquired immune deficiency syndrome|
|US4704354 *||Feb 15, 1984||Nov 3, 1987||University Of Cincinnati||Virion assay method for use in in vitro screening of teratogens and carcinogens|
|US4704355 *||Mar 27, 1985||Nov 3, 1987||New Horizons Diagnostics Corporation||Assay utilizing ATP encapsulated within liposome particles|
|US4727019 *||May 10, 1985||Feb 23, 1988||Hybritech Incorporated||Method and apparatus for immunoassays|
|US4729961 *||Dec 11, 1985||Mar 8, 1988||Institut Pasteur||Process for the detection and assay by erythroadsorption|
|US4737453 *||Dec 12, 1984||Apr 12, 1988||Immunomedics, Inc.||Sandwich immunoassay utilizing a separation specific binding substance|
|US4740456 *||Aug 15, 1985||Apr 26, 1988||Wake Forest University||Immunological methods for diagnosing neurocysticercosis|
|US4772550 *||Feb 10, 1986||Sep 20, 1988||Miles Inc.||Heterogeneous specific binding assay employing an aggregatable binding reagent|
|US4801532 *||Sep 30, 1987||Jan 31, 1989||Wake Forest University||Immunological methods for diagnosing neurocysticercosis|
|US4806546 *||Sep 30, 1985||Feb 21, 1989||Miles Inc.||Immobilization of nucleic acids on derivatized nylon supports|
|US4806631 *||Sep 30, 1985||Feb 21, 1989||Miles Inc.||Immobilization of nucleic acids on solvolyzed nylon supports|
|US4849505 *||Apr 28, 1987||Jul 18, 1989||Enzo Biochem, Inc.||Detectable molecules, method of preparation and use|
|US4870007 *||Dec 18, 1987||Sep 26, 1989||Eastman Kodak Company||Immobilized biotinylated receptor in test device, kit and method for determining a ligand|
|US4889798 *||Feb 13, 1987||Dec 26, 1989||Enzo Biochem, Inc.||Heterologous system for the detection of chemically labeled DNA and other biological materials providing a receptor or target moiety thereon|
|US4894325 *||Jan 15, 1987||Jan 16, 1990||Enzo Biochem, Inc.||Hybridization method for the detection of genetic material|
|US4933275 *||Oct 24, 1985||Jun 12, 1990||The General Hospital Corporation||Method for the detection of a polypeptide subunit in the presence of a quaternary protein containing the subunit|
|US4935339 *||May 7, 1985||Jun 19, 1990||Nichols Institute Diagnostics||Delayed solid phase immunologic assay|
|US4943523 *||Apr 28, 1987||Jul 24, 1990||Enzo Biochem, Inc.||Detectable molecules, method of preparation and use|
|US4959307 *||Sep 5, 1986||Sep 25, 1990||Syntex (U.S.A.) Inc.||Immunoseparating strip|
|US5001048 *||Jun 5, 1987||Mar 19, 1991||Aurthur D. Little, Inc.||Electrical biosensor containing a biological receptor immobilized and stabilized in a protein film|
|US5002885 *||Apr 24, 1990||Mar 26, 1991||Enzo Biochem, Inc.||Detectable molecules, method preparation and use|
|US5028535 *||Jan 10, 1989||Jul 2, 1991||Biosite Diagnostics, Inc.||Threshold ligand-receptor assay|
|US5085988 *||Jul 5, 1990||Feb 4, 1992||Syntex (U.S.A.) Inc.||Immunoseparating strip|
|US5143852 *||Sep 14, 1990||Sep 1, 1992||Biosite Diagnostics, Inc.||Antibodies to ligand analogues and their utility in ligand-receptor assays|
|US5168057 *||Sep 30, 1991||Dec 1, 1992||Beckman Instruments, Inc.||Trifunctional conjugates|
|US5180828 *||Apr 9, 1992||Jan 19, 1993||Molecular Devices Corporation||Chromophoric reagents for incorporation of biotin or other haptens into macromolecules|
|US5192507 *||Mar 4, 1991||Mar 9, 1993||Arthur D. Little, Inc.||Receptor-based biosensors|
|US5196351 *||Jun 8, 1990||Mar 23, 1993||Beckman Instruments, Inc.||Bidentate conjugate and method of use thereof|
|US5200313 *||Apr 25, 1988||Apr 6, 1993||Miles Inc.||Nucleic acid hybridization assay employing detectable anti-hybrid antibodies|
|US5225353 *||Oct 3, 1991||Jul 6, 1993||Fred Hutchinson Cancer Research Center||Method for immunoselection of cells using avidin and biotin|
|US5262334 *||Oct 3, 1991||Nov 16, 1993||Fred Hutchinson Cancer Research Center||Method for immunoselection of cells using avidin and biotin|
|US5332679 *||Feb 12, 1993||Jul 26, 1994||E. I. Dupont De Nemours And Company||Method for specific binding assays using a releasable ligand|
|US5366864 *||Sep 30, 1992||Nov 22, 1994||Eastman Kodak Company||Buffered wash composition, insolubilizing composition, test kits and method of use|
|US5374516 *||Feb 24, 1989||Dec 20, 1994||Eastman Kodak Company||Avidin-and biotin immobilized reagents and methods of use|
|US5378608 *||Jun 22, 1990||Jan 3, 1995||Nippon Shoji Kabushiki Kaisha||Enzyme immunoassay for antigen and solid phase used therefor|
|US5422281 *||Jul 31, 1992||Jun 6, 1995||Beckman Instruments, Inc.||Bidentate conjugate and method of use thereof|
|US5437983 *||Feb 1, 1993||Aug 1, 1995||Syntex (U.S.A.) Inc.||Heterogeneous binding assays|
|US5459078 *||Jun 9, 1993||Oct 17, 1995||Abbott Laboratories||Methods and reagents for performing ion-capture digoxin assays|
|US5459080 *||Jan 27, 1994||Oct 17, 1995||Abbott Laboratories||Ion-capture assays using a specific binding member conjugated to carboxymethylamylose|
|US5501949 *||Aug 15, 1994||Mar 26, 1996||Murex Diagnostics Corporation||Particle bound binding component immunoassay|
|US5512492 *||May 18, 1993||Apr 30, 1996||University Of Utah Research Foundation||Waveguide immunosensor with coating chemistry providing enhanced sensitivity|
|US5512659 *||Aug 4, 1989||Apr 30, 1996||Syntex (U.S.A.) Inc.||Compositions useful in heterogeneous immunoassays|
|US5518882 *||Dec 21, 1993||May 21, 1996||Biotex Laboratories, Inc.||Immunological methods of component selection and recovery|
|US5534620 *||May 25, 1995||Jul 9, 1996||Beckman Instruments, Inc.||Method of heterogenous purification using a bidentate conjugate|
|US5536820 *||Jun 23, 1994||Jul 16, 1996||E. I. Du Pont De Nemours And Company||Avidin-binding azo reagents|
|US5612221 *||May 30, 1995||Mar 18, 1997||Dade Chemistry Systems Inc.||Avidin-binding fluorescing and quenching reagent for use in homogeneous assays|
|US5661019 *||Mar 22, 1995||Aug 26, 1997||Beckman Instruments, Inc.||Trifunctional conjugates|
|US5677196 *||Jun 22, 1994||Oct 14, 1997||University Of Utah Research Foundation||Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays|
|US5679526 *||Aug 1, 1994||Oct 21, 1997||Biosite Diagnostics Incorporated||Threshold ligand-receptor assay|
|US5700639 *||Jun 9, 1994||Dec 23, 1997||Boehringer Mannheim Gmbh||Method for the detection of metabolically labelled DNA|
|US5726010 *||Jul 31, 1991||Mar 10, 1998||Idexx Laboratories, Inc.||Reversible flow chromatographic binding assay|
|US5726013 *||Jun 7, 1995||Mar 10, 1998||Idexx Laboratories, Inc.||Reversible flow chromatographic binding assay system, kit, and method|
|US5747352 *||May 23, 1994||May 5, 1998||Beckman Instruments, Inc.||Reagents and methods for the rapid and quantitative assay of pharmacological agents|
|US5750333 *||Jun 7, 1995||May 12, 1998||Idexx Laboratories, Inc.||Reversible flow chromatographic binding assay|
|US5773224 *||Feb 12, 1996||Jun 30, 1998||Grandics; Peter||Immunoselection system for cell elution|
|US5776702 *||Jul 7, 1994||Jul 7, 1998||Boehringer Mannheim Gmbh||Process for the determination of an immunologically detectable substance and a suitable reaction vessel therefor|
|US5814565 *||Feb 23, 1995||Sep 29, 1998||University Of Utah Research Foundation||Integrated optic waveguide immunosensor|
|US5846842 *||Apr 30, 1996||Dec 8, 1998||University Of Utah Research Foundation||Waveguide immunosensor with coating chemistry and providing enhanced sensitivity|
|US5866322 *||Oct 11, 1991||Feb 2, 1999||Abbott Laboratories||Method for performing Rubella assay|
|US5877028||Mar 31, 1993||Mar 2, 1999||Smithkline Diagnostics, Inc.||Immunochromatographic assay device|
|US5919712 *||Nov 13, 1996||Jul 6, 1999||University Of Utah Research Foundation||Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays|
|US5935780 *||Sep 21, 1995||Aug 10, 1999||Boehringer Mannheim Gmbh||Method for the qualitative or/and quantitative detection of an analyte|
|US5939272 *||Jun 11, 1997||Aug 17, 1999||Biosite Diagnostics Incorporated||Non-competitive threshold ligand-receptor assays|
|US5998220||Feb 10, 1994||Dec 7, 1999||Beckman Coulter, Inc.||Opposable-element assay devices, kits, and methods employing them|
|US6007999 *||Mar 9, 1998||Dec 28, 1999||Idexx Laboratories, Inc.||Reversible flow chromatographic binding assay|
|US6017767||Jun 5, 1995||Jan 25, 2000||Beckman Coulter, Inc.||Assay device|
|US6030846 *||Jan 10, 1997||Feb 29, 2000||Dade Behring Inc.||Homogeneous assays using avidin-binding azo reagents|
|US6140138 *||May 4, 1994||Oct 31, 2000||Igen International Inc.||Electrochemiluminescent metal chelates and means for detection|
|US6153442 *||May 20, 1998||Nov 28, 2000||Dade Behring Inc.||Reagents and methods for specific binding assays|
|US6168956||May 29, 1991||Jan 2, 2001||Beckman Coulter, Inc.||Multiple component chromatographic assay device|
|US6210978 *||Mar 2, 1998||Apr 3, 2001||Bayer Corporation||Method and device for the detection of analyte in a fluid test sample|
|US6222619||Sep 18, 1997||Apr 24, 2001||University Of Utah Research Foundation||Diagnostic device and method|
|US6291169||Mar 30, 1999||Sep 18, 2001||Molecular Devices Corporation||Hapten derivatized capture membrane and diagnostic assays using such membrane|
|US6297060||Feb 11, 1999||Oct 2, 2001||Biosite Diagnostics, Inc.||Assay devices comprising a porous capture membrane in fluid-withdrawing contact with a nonabsorbent capillary network|
|US6316274||Mar 1, 2000||Nov 13, 2001||University Of Utah Research Foundation||Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays|
|US6326136||Dec 23, 1994||Dec 4, 2001||Digene Corporation||Macromolecular conjugate made using unsaturated aldehydes|
|US6340598||Dec 8, 1998||Jan 22, 2002||University Of Utah Research Foundation||Apparatus for multichannel fluorescent immunoassays|
|US6489148 *||May 12, 2000||Dec 3, 2002||Board Of Trustees Of Michigan State University||Immunoassay for equine protozoal myeloencephalitis in horses|
|US6534323 *||Jan 21, 2000||Mar 18, 2003||Medlyte Diagnostics, Inc.||Compositions and methods for early detection of heart disease|
|US6656744||Feb 22, 2000||Dec 2, 2003||Quidel Corporation||One-step lateral flow assays|
|US6979567||Nov 13, 2001||Dec 27, 2005||Biocentrex, Llc||Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays|
|US7022515||Oct 5, 2001||Apr 4, 2006||University Of Utah Research Foundation||Waveguide immunosensor with coating chemistry and providing enhanced sensitivity|
|US7087420||Mar 16, 2000||Aug 8, 2006||Cambia||Microbial β-glucuronidase genes, gene products and uses thereof|
|US7105311 *||May 3, 2002||Sep 12, 2006||Immunetics, Inc.||Systems and methods for detection of analytes in biological fluids|
|US7125517||Aug 9, 2004||Oct 24, 2006||Immunetics, Inc.||Systems and methods for detection of analytes in biological fluids|
|US7141719||Apr 11, 2002||Nov 28, 2006||Cambia||Microbial β-Glucuronidase genes, gene production and uses thereof|
|US7262019||May 24, 2004||Aug 28, 2007||Immunetics, Inc.||System and methods for detection of Bacillus anthracis related analytes in biological fluids|
|US7713528||Jun 7, 1995||May 11, 2010||Enzo Therapeutics, Inc.||Method for in vivo delivery of active compounds using reagent conjugate|
|US20020160534 *||Oct 5, 2001||Oct 31, 2002||Herron James N.||Waveguide immunosensor with coating chemistry and providing enhanced sensitivity|
|US20030157684 *||Apr 11, 2002||Aug 21, 2003||Cambia||Microbial B-glucuronidase genes, gene production and uses thereof|
|US20030185870 *||Nov 20, 2002||Oct 2, 2003||Grinstaff Mark W.||Interfacial biomaterials|
|US20050042696 *||Aug 9, 2004||Feb 24, 2005||Victor Kovalenko||Systems and methods for detection of analytes in biological fluids|
|US20070134739 *||Dec 12, 2005||Jun 14, 2007||Gyros Patent Ab||Microfluidic assays and microfluidic devices|
|US20070148707 *||Aug 25, 2006||Jun 28, 2007||Immunetics, Inc.||Systems and methods for detection of analytes in biological fluids|
|US20070178542 *||May 24, 2004||Aug 2, 2007||Immunetics, Inc.||System and methods for detection of bacillus anthracis related analytes in biological fluids|
|US20110116972 *||Oct 14, 2010||May 19, 2011||Mats Holmquist||Microfluidic assays and microfluidic devices|
|US20170160294 *||May 11, 2016||Jun 8, 2017||Life Technologies As||Methods of reversibly binding a biotin compound to a support|
|EP0170652A1 *||Jan 27, 1984||Feb 12, 1986||Molecular Biosystems, Inc.||Assay for immobilized reporter groups|
|EP0170652A4 *||Jan 27, 1984||Aug 23, 1988||Molecular Biosystems Inc||Assay for immobilized reporter groups.|
|EP0180638A1 *||May 13, 1985||May 14, 1986||Hybritech Inc||Method and apparatus for immunoassays.|
|EP0180638A4 *||May 13, 1985||Sep 28, 1988||Hybritech Inc||Method and apparatus for immunoassays.|
|EP0232816A2 *||Jan 30, 1987||Aug 19, 1987||Miles Inc.||Heterogeneous specific binding assay employing an aggregatable binding reagent|
|EP0232816A3 *||Jan 30, 1987||Nov 9, 1988||Miles Inc.||Heterogeneous specific binding assay employing an aggregatable binding reagent|
|EP0310872A1 *||Sep 22, 1988||Apr 12, 1989||Hygeia Sciences, Inc.||Metal sol capture immunoassay procedure, kit for use therewith and captured metal containing composite|
|EP0475783A1||Sep 13, 1991||Mar 18, 1992||Biosite Diagnostics Inc.||Antibodies to ligand analogues and their utility in ligand-receptor assays|
|EP1546301A2 *||Jul 10, 2003||Jun 29, 2005||Steve Berstein||Device and methods for isolating target cells|
|EP1546301A4 *||Jul 10, 2003||Dec 20, 2006||Nexell Of California Inc||Device and methods for isolating target cells|
|EP2237037A1||Dec 12, 2005||Oct 6, 2010||Gyros Patent Ab||Microfluidic device and use thereof|
|EP2428226A1||Oct 22, 2002||Mar 14, 2012||The Scripps Research Institute||Antibody targeting compounds|
|WO1983002285A1 *||Dec 20, 1982||Jul 7, 1983||Boston Biomed Res Inst||Bispecific antibody determinants|
|WO1984004171A1 *||Dec 14, 1983||Oct 25, 1984||Quidel||Detection of human chorionic gonadotropin|
|WO1985003356A1 *||Jan 27, 1984||Aug 1, 1985||Molecular Biosystems, Inc.||Assay for immobilized reporter groups|
|WO1985005451A1 *||May 13, 1985||Dec 5, 1985||Hybritech Incorporated||Method and apparatus for immunoassays|
|WO1987004794A1 *||Feb 4, 1987||Aug 13, 1987||Microbiological Associates, Inc.||Latex agglutination using avidin/biotin system|
|WO1990004786A1||Oct 10, 1989||May 3, 1990||Molecular Devices Corporation||Hapten derivatized capture membrane and diagnostic assays using such membrane|
|WO1990006670A2 *||Dec 6, 1989||Jun 28, 1990||The General Hospital Corporation||Method of enrichment and cloning for dna containing an insertion or corresponding to a deletion|
|WO1990006670A3 *||Dec 6, 1989||Jul 26, 1990||Gen Hospital Corp||Method of enrichment and cloning for dna containing an insertion or corresponding to a deletion|
|WO1995021942A1 *||Feb 6, 1995||Aug 17, 1995||T Cell Diagnostics, Inc.||Immunoassay kit comprising universal reagents|
|WO1997006439A1 *||Aug 9, 1996||Feb 20, 1997||Quidel Corporation||Test strip and method for one step lateral flow assay|
|WO2004027021A2 *||Jul 10, 2003||Apr 1, 2004||Nexell Of California, Inc.||Device and methods for isolating target cells|
|WO2004027021A3 *||Jul 10, 2003||Jul 8, 2004||Steve Berstein||Device and methods for isolating target cells|
|U.S. Classification||435/7.5, 436/529, 435/810, 435/975, 436/530, 436/808, 436/544, 435/7.93, 436/547, 435/188, 436/527, 436/531|
|International Classification||C12Q1/32, G01N33/53, C12Q1/533, G01N33/535, C12Q1/42, G01N33/537, C12Q1/28, G01N33/543, G01N33/538|
|Cooperative Classification||G01N33/538, Y10S435/975, Y10S436/808, Y10S435/81, G01N33/535, G01N33/537|
|European Classification||G01N33/535, G01N33/538, G01N33/537|
|Feb 18, 1992||AS||Assignment|
Owner name: BCW-USA, INC.
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:BURROUGHS WELLCOME CO.;REEL/FRAME:006014/0767
Effective date: 19920207
|Jun 7, 1993||AS||Assignment|
Owner name: MUREX DIAGNOSTICS, INC., GEORGIA
Free format text: CHANGE OF NAME;ASSIGNOR:BCW-USA, INC.;REEL/FRAME:006576/0514
Effective date: 19920429